Process for the isolation of lovastatin

ABSTRACT

The process for the preparation and isolation of the hypolipaemic active substance lovastatin in substantially pure form having a purity of at least 95% which comprises lactonizing the mevinolinic acid to lovastatin in a totally aqueous medium.

FIELD OF THE INVENTION

The present invention relates to a process for the preparation andisolation of the hypolipaemic active substance lovastatin insubstantially pure form having a purity of at least 95% which compriseslactonizing the mevinolinic acid of Formula II

to lovastatin of Formula I

in a totally aqueous medium.

BACKGROUND OF THE INVENTION

It is known that certain mevalonate derivatives are active asanti-hyper-cholesterolemic agents, and these function by limitingcholesterol biosynthesis by inhibiting the enzyme HMG-CoA reductase.Lovastatin, Pravastatin, Simvastatin, Mevastatin, Atorvastatin andderivatives and analogs thereof are known as HMG-COA reductaseinhibitors and are used as anti-hypercholesterolemic agents. Themajority of them are produced by fermentation using microorganisms ofdifferent species belonging to Aspergillus, Monascus, Nocardia,Amycolatopsis, Mucor or Penicillium genus; others are obtained bytreating the fermentation products using the method of chemicalsynthesis or they are the product of total chemical synthesis.

Lovastatin is the first of the statins to be used widely and ismanufactured by a fermentation-based process. It is produced as asecondary metabolite of the fungus Aspergillus terreus (U.S. Pat. No.4,231,938) deposited in American Type Culture Collection under Nos. ATCC20541 and ATCC 20542, and Monascus ruber deposited in FermentationResearch Institute Agency of Industrial Science and Technology, Ministryof International Trade and Industry, Japan (DE 3006216 Al) under No.Ferm. 4822.

Chemically lovastatin is1,2,6,7,8,8a-hexahydro-β,δ-dihydroxy-2,6-dimethyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoic acid δ-lactone of Formula I:

An active form of lovastatin is an acid, which is chemically1,2,6,7,8,8a-hexahydro-β,δ-dihydroxy-2,6-dimethyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoic acid of Formula II:

The recent commercial introduction of chemically synthesised HMG-CoAreductase inhibitors has provided a need for the development of highyielding processes for production of fermentation-based statins. Thetechniques to improve the processes include, but are not limited to,improving the producer microorganism, scale-up of the process, improvingthe culture medium or simplifying the downstream recovery process.

In the fermentation broth, lovastatin is present mostly in its hydroxyacid form, mevinolinic acid. The isolation of lovastatin from thefermentation broth, can be categorised into two routes in the prior artprocesses. The first route comprises of solvent extraction ofmevinolinic acid and isolation of ammonium salt of mevinolinic acid asan intermediate and its further lactonization to lovastatin (U.S. Pat.No. 4,319,039).

The second route comprises of solvent extraction of mevinolinic acid,lactonization in the solvent phase and isolation as lovastatin (PCTpublication WO 97/20834). The isolation of lovastatin as disclosed in EP033536 comprises of extraction of the broth with ethyl acetate. Theextract is concentrated by vacuum distillation followed by lactonizationin toluene at 106° C. for 2 hours. After the lactonization is complete,the solution is concentrated to a small volume and then subjected tocolumn chromatography using solvents like ethyl acetate or n-hexane andthe collected fractions are again concentrated in vacuum and then purelovastatin crystallizes in the lactone form.

Both the routes may employ a final purification step to obtainlovastatin of pharmacopoeial grade.

The process for the isolation and purification ofanti-hypercholesterolemic agents disclosed in the earlier patents havecertain inherent disadvantages and involves a number of steps whichinclude multiple solvent extractions, chromatography, lactonization andcrystallization methods. Although, the purity of the final productobtained by these procedures is of pharmacopoeial standards yet, theyields of the desired product are relatively low. In addition, theyrequire both large amounts of organic solvents and correspondingly largeequipment suited for these quantities. The second route involveslactonization at higher temperature in the solvent phase necessitatingelaborate purification step(s) for removal of undesirable impuritiesgenerated during the lactonization step.

Neither of the methods heretofore described is completely satisfactoryfor the above-stated reasons.

SUMMARY OF THE INVENTION

It is an object of the present invention to solve the problemsassociated with the prior art and to provide a simplified and efficientmethod for the preparation of lovastatin, leading to aneconomically-more attractive method of manufacturing. The process, inaddition, also yields higher quality product.

The present invention solves the drawbacks of the processes known in theprior art as it enables to obtain the pure lovastatin by a process,which process is less time consuming and provides higher yields usingfewer number of solvents. The process is more nature friendly, is notdemanding in terms of space and energy and thus, enables an economicallarge scale production.

The present invention specifically describes a process for effecting thelactonization of mevinolinic acid of Formula II

in the aqueous medium and isolating the corresponding lovastatin ofFormula I:

in substantially pure form from the solution thereof. The lactonizationis effected in a totally aqueous medium, the fermentation broth itself.Lovastatin, so obtained is in substantially pure form and has achromatographic purity of at least 95%.

According to the present invention, lovastatin is derived from thefermentation broth which comprises of the microorganism preferably afungus belonging to the genus Aspergillus, preferably Aspergillusterreus (ATCC 20542) or its hyperproducer thereof.

The process comprises lowering the pH of the fermented broth containinglovastatin, present mostly in the form of mevinolinic acid to 2.0 to3.0, and incubating the broth for about 20–60 hours at a temperaturefrom about 40–60° C. affecting the lactonization of mevinolinic acid andrecovering the corresponding lovastatin from the solution thereof. Theacid for lowering the pH may be selected from the commonly used acids,preferably the mineral acids like hydrochloric acid, sulfuric acid,nitric acid, phosphoric acid, etc.

In a preferred embodiment, the broth is incubated for 24 hours. In afurther preferred embodiment, the broth is incubated at a temperature inthe range of 40–60° C., most preferable being 50–60° C.

The subsequent isolation of lovastatin is carried out by conventionalmethods known in the art to a person of ordinary skill. Lovastatin isisolated from the solution by extracting it with a solvent followed byconcentration of solvent, crystallization and drying the product.Solvent which may be used is generally selected from esters such asethyl acetate, butyl acetate or aromatic hydrocarbons such as toluene.Methods known in the art may be used with the process of this inventionto enhance any aspect of this process. For example, lovastatin obtainedmay further be purified. The purification can be such asrecrystallization from solvents.

DETAILED DESCRIPTION OF THE INVENTION

The following specific examples illustrate the process of presentinvention, but they are not intended to limit the scope of theinvention.

EXAMPLE 1

To 6.2 L of fermentation broth, 0.80 L of dilute sulfuric acid (2N) wasadded to bring the pH to 2.1. The acidified broth was heated to about50° C. and was stirred at 50–60° C. for about 24 hours. 90% of the acidform was converted to lactone form as monitored by High Pressure LiquidChromatography.

The broth was filtered and the mycelial cake was extracted with 7.5 L oftoluene. The toluene extract was washed with 1.3 L of 5% w/v sodiumbicarbonate solution and 0.65 L of demineralised water. The washedtoluene extract was concentrated under vacuum at about 60° C., to avolume of about 160–200 mL. The concentrate was cooled to 5–7° C. andstirred further for 1 hour. The slurry was then filtered and the cakewas washed with 50 mL of pre-cooled (5–10° C.) toluene. The wet cake wasdried at 40° C. under vacuum for 16 hours. 25.6 g of Lovastatin with achromatographic purity of more than 95% was obtained.

EXAMPLE 2

To 12 L of fermentation broth, 1.40 L of dilute nitric acid (10%) wasadded to bring the pH to 2.2. The acidified broth was heated to about50° C. and was stirred at this temperature for about 48 hours. 90% ofthe acid form was converted to lactone form as monitored by HighPressure Liquid Chromatography.

The broth was filtered and the mycelial cake was extracted with 12 L oftoluene. The toluene extract was washed with 1.5 L of 5% w/v sodiumbicarbonate solution and 1.5 L of demineralised water. The washedtoluene extract was concentrated under vacuum at about 50° C., to avolume of about 300–350 mL. The concentrate was cooled to 5–7° C. andstirred at this temperature for 3 hours. The slurry was then filteredand the cake was washed with 100 mL of pre-cooled (5–10° C.) toluene.The wet cake was dried at 40° C. under vacuum. 46.2 g of Lovastatin witha chromatographic purity of more than 95% was obtained.

EXAMPLE 3

To 8500 L of fermentation broth, 540 L of dilute sulfuric acid (2N) wasadded to bring the pH to 2.0. The acidified broth was heated to 50–55°C. and was stirred further for about 22 hours. More than 90% of the acidform was converted to lactone form, as monitored by High Pressure LiquidChromatography.

The whole broth was extracted with 7600 L of toluene. The tolueneextract was washed with 1340 L of 5% w/v sodium bicarbonate solution and670 L of demineralised water. The washed toluene extract wasconcentrated under vacuum at 40–60° C., to a volume of approximately 200L. The concentrate was cooled to 5–8° C. and stirred further for 2hours. The slurry was then filtered and the cake was washed with 130 Lof pre-cooled (5–10° C.) toluene. The wet cake was dried to yield 45.3Kg of Lovastatin with a chromatographic purity of more than 95%.

While the present invention has been described in terms of its specificembodiments, certain modifications and equivalents will be apparent tothose skilled in the art and are intended to be included within thescope of the present invention.

1. A process for effecting the lactonization of mevinolinic acid ofFormula II

in an aqueous medium, the process lowering the pH of a fermentationbroth and isolating the corresponding lovastatin of Formula I soobtained

in substantially pure form.
 2. The process of claim 1, wherein thefermentation broth is incubated at a temperature of from about 40–60° C.3. The process of claim 2, wherein the fermentation broth comprises ofthe microorganism capable of producing lovastatin.
 4. The process ofclaim 3, wherein the microorganism is a fungus.
 5. The process of claim4, wherein the fungus belongs to the genus Aspergillus.
 6. The processof claim 5, wherein the fungus is Aspergillus terreus (ATCC 20542). 7.The process of claim 1, wherein the pH of the fermentation broth islowered with an acid in the range of 2.0–3.0.
 8. The process of claim 7,wherein the acid is a mineral acid.
 9. The process of claim 8, whereinthe mineral acid is selected from the group consisting of sulfuric acid,nitric acid, hydrochloric acid or phosphoric acid.
 10. The process ofclaim 1, which further comprises isolating the lovastatin from thefermentation broth by extracting with a solvent followed byconcentration of solvent extract, crystallization and drying oflovastatin.
 11. The process of claim 10, wherein the solvent is selectedfrom esters such as ethyl acetate, butyl acetate or aromatichydrocarbons such as toluene.